There exists much current effort and research being expended toward the genetic transformation of plant species. It is believed that the development of efficient means for transforming foreign genes into plant germ lines will allow the diversity of the genetic stock in commercially important crop species to be widened and to allow functional genes of specific interest to be selectively introduced into crop species. The effort and research to date on the transformation, or genetic engineering, of plant species has achieved results which vary quite dramatically depending on the species of plant.
The principal mechanism which has been used heretofore for the introduction of exogenous genes into plants has begun with the transformation of single plant cells either as protoplasts or in an undifferentiated tissue mass known as a callus. Chimeric genes functional in plant cells have been introduced into single cell plant protoplasts by electroporation and microinjection. However, the most widely used transformation technique used to date has taken advantage of a natural trait of the Plant Pathogen Agrobacterium tumefaciens, which has the innate ability to transfer a portion of the DNA from a Ti (Tumor-inducing) plasmid harbored in it into an infected plant cell. By inserting foreign genes into plasmids in Agrobacterium which carry certain sequences from the Ti plasmid, the bacterial transformational trait can be used to transport the foreign genes into the genome of the infected plant cells. Agrobacterium-mediated plant cell transformation has been found to work reasonably well in many model crop species, such as tobacco, petunia and carrot, but does suffer from two significant limitations. The first limitation is that the mediation can only be done on an individual cellular level, typically with somatic tissues, which then must be regenerated artificially into a whole plant. This limits the applicability of Agrobacterium-mediated genetic transformation to those crop species which can readily be regenerated from types of tissues which are susceptible to Agrobacterium infection. A second limitation is that the natural host range of Agrobacterium includes only dicotyledonous plants and a limited number of monocot species of the Liliaceae family. Therefore Agrobacterium-mediated transformation has not been proven to be an effective tool for monocot species of commercial interests, such as the cereal crop species.
It has been demonstrated that at least some chimeric gene constructions are effective for expression of foreign genes in most plant cells. The functionality of these chimeric constructions in monocots as well as dicots has been demonstrated by the transformation of maize protoplasts in culture through such techniques as electroporation. However, no currently known methodology exists to regenerate whole maize plants, or whole plants of any other important crop species, from such protoplasts. No whole, intact transformed maize plants, for example, are known to have been generated. Nevertheless genetic transformation of lines of maize and other crop species is a desired objective because of the great agricultural value of the common crop plants and the potential to improve their value and productivity.
There has been at least one suggestion previously that maize plants can be genetically transformed by genetic transformation of their pollen. Published PCT patent application WO 85/01856 to DeWet purportedly describes a method for the transfer of exogenous genes into flowering plants by transforming the pollen of the plants. Attempts by others to verify this technique and reproduce the experiment have failed. Sanford et al., Theor. Appl. Genet., 69 (5-6), 571-74 (1985). A report of one similar result has been made. Ohta, Proc. Natl. Acad. Sci. USA, 83:715-719 (1986).